The mechanism of action of 3,6-bis(2-piperidinoethoxy)acridine trihydrochloride (CL 246,738) in the potentiation of natural killer cells

An effort has been made to determine the mechanism by which the immunomodulator 3,6-bis(2-piperidinoethoxy)acridine trihydrochloride (CL 246,738) enhances the cytotoxic activity of natural killer (NK) cells. Orally administered CL 246,738 produced augmentation of NK cell activity in mice in a dose-related fashion over a dose range of 10 to 160 mg/kg, with a peak stimulation occurring at 40 mg/kg. The stimulatory effect was short-lived and only persisted for 3 days after a single oral dose of the drug. However, it could be boosted by a subsequent treatment. With anti-asialo GM-1 (anti-ASGM-1) antibody used as an NK cell marker, it was determined that the compound increased the number of ASGM-1-positive cells in mice, as indicated by radioimmunoassay and immunofluorescence staining. NK cells of beige mice were also activated by CL 246,738. Furthermore, the compound at concentrations of 0.02 to 0.2 microgram/ml induced NK cell activity in vitro, with a minimum 3-day incubation being required for optimal activation. This effect was dependent on the presence of macrophages and was inhibited by anti-IFN-alpha + beta but not anti-IFN-beta antibody. Taken together, it is postulated that the compound functions by stimulating macrophages to release IFN-alpha, which subsequently activates NK cells. As an effective stimulator of IFN and NK cells, CL 246,738 may prove clinically useful in the immunotherapy of certain types of malignancy.     

Reaction of 1,3-bis(2-chloroethyl)-1-nitrosourea with synthetic polynucleotides.

The antitumor agent BCNU (1,3-bis(2-chloroethyl)-1-nitrosourea) was incubated with poly(C) and poly(G) in aqueous solution at 37 degrees and pH 7 to produce approximately 0.33 and 0.07% substitution, respectively. Under the same conditions, there was relatively little reaction with poly(A) and poly(U). Poylnucleotides reacted with [14C]BCNU were digested by chemical and enzymatic methods, and the derivative nucleotides were isolated by column chromatography. There were identified by a combination of ultraviolet and mass spectroscopy as 3-(beta-hydroxyethyl)CMP, 3,N4-ethano-CMP, and 7-(beta-hydroxy-ethyl)GMP. This would indicate that BCNU generates active two carbon fragments, probably chloroethyl carbonium ions, which are free to react with nucleotides. The production of these substituted bases may be important to the mechanism of action of the therapeutic nitrosoureas since they would probably alter the function of any nucleic acid which contained them.     

Inhibition of the growth of peas by tris-(2-diethylaminoethyl)-phosphate trihydrochloride

The effects of Tris-(2-diethylaminoethyl)-phosphate trihydrochloride (SK&F 7997-A₃) on the development of 4 varieties of Pisum sativum were investigated. The compound inhibited shoot elongation of all 4 varieties by as much as 50% or more when seeds were soaked in solutions of the inhibitor for 12 hours before planting. Seed treatment also affected flowering by causing an increase in the number of nodes to the first flower in the early varieties Little Marvel and Alaska. The number of nodes preceding the first flower in the late varieties Dwarf and Tall Telephone was not affected by high concentrations of SK&F 7997-A₃, but low concentrations appeared to cause a slight reduction in the number of nodes to flower.

The inhibitor had little effect on growth when applied to established seedlings; some slight inhibition was noted when high doses were applied to the shoot tip.

SK&F 7997-A₃ suppressed the growth response of dwarf and tall peas to exogenous GA₃. The compound did not inhibit biosynthesis of gibberellin by Fusarium moniliformc when present in shaken liquid cultures at concentrations as high as 10 mg/ml. The inhibitor suppressed the action of applied GA₃ on shoot elongation when the 2 chemicals were applied in 3 ways: 1) inhibitor on lowermost compound leaf and GA₃ on shoot tip; 2) GA₃ on lowermost leaf and inhibitor on shoot tip; and 3) soaking of seeds in the 2 compounds combined for 12 hours prior to planting. The third method of dual treatment yielded evidence that SK&F 7997-A₃ interacts noncompetitively with GA₃ in the regulation of shoot elongation.

     

TH1 and TH2 cytokine inhibition by 3,5-bis(trifluoromethyl)pyrazoles,a novel class of immunomodulators

In order to discover novel immunomodulators for application in treating autoimmune diseases, a stable Jurkat transfectant was constructed in which luciferase reporter gene is driven by a full-length IL-2 promotor. A chemical library was screened to identify compounds that inhibited luciferase expression in Jurkat transfectants stimulated with PMA and ionomycin. A class of compounds (bis-trifluoromethyl pyrazole, BTPs) was identified from this screen. BTPs were shown to inhibit anti-CD3 and anti-CD28 antibody-induced IL-2 secretion, mixed lymphocyte reaction, and Con A-induced T cell proliferation in normal human peripheral blood T cells. In addition, mRNA levels of IL-4, IL-5, IL-9, IL-10, IL-13, IL-15, and IFN-gamma were markedly inhibited by BTPs in peripheral blood mononuclear cells stimulated by Con A as determined by multi-probe RNA protection assay. Furthermore, IL-2, IL-4, IL-5, and IFN-gamma secretion by Hut 78 cells or CD3(+) T cells stimulated with PMA plus ionomycin or anti-CD3 antibody plus PMA were inhibited in a concentration-dependent manner by BTPs. Therefore, BTPs inhibit a wide spectrum of cytokine production including TH1 and TH2 type cytokines. Taken together, these compounds may be useful for treating autoimmune diseases and organ transplant rejection.     

Modulation of the immune response to tumors by a novel synthetic compound,N-[4-[(4-fluorophenyl)sulfonyl]phenyl] acetamide (CL 259,763)

Summary CL 259,763, N-[4-[(4-fluorophenyl)sulfonyl]phenyl] acetamide, is an orally active compound capable of modifying the reactivity of certain lymphoid cell populations affected by the growth of a tumor. The compound augmented the response of lymphocytes from tumor-primed animals to syngeneic tumor cells, resulting in a marked increase in tumor cell destruction. Likewise, it enhanced macrophage inhibitory effects on the growth of tumor cells in vitro. These activated macrophages were detectable in peritoneal exudates of treated mice 4 to 12 days after receiving a single oral dose of CL 259,763, with peak activity being demonstrable by day 7. The compound also restored the alloreactivity of lymphocytes from immunodepressed mice bearing the Lieberman plasma cell tumor, possibly by interferring with suppressor cells. Macrophages and lymphocytes from treated mice released significantly more IL-1 and IL-2-like factors in culture than did the control counterparts. Sera from treated mice also possessed more colony stimulating factor than those from normal mice. Immunoadjuvant effects were evident when the compound was administered with an inactivated L1210 leukemia vaccine and it enhanced the effectiveness of cytotoxic chemotherapy when given to mice challenged with P388 murine leukemia. These immunomodulating effects of CL 259,763 may hopefully be exploited in efforts to augment the immune response of the host to a progressively growing tumor.     

Induction of cytoplasmically inherited respiration-deficient ('petite') mutants by photodynamic action of acridine compounds

All acridines used (acriflavine, proflavine, acridine orange and 3-azido-10-methylacridinium chloride) produced killing in yeast cells when activated with visible light. Acriflavine, proflavine and 3-azido-10-methylacridinium chloride, but not acridine orange, produced petite and sectored colonies. Both cell killing and petite induction by light activation of acriflavine resulted apparently from photodynamic action mediated by singlet oxygen (1O2) since the effect were prevented by either sodium azide or anaerobiosis. The biological effects of 3-azido-10-methylacridinium chloride, which was developed as a potential photoaffinity probe for studying the binding and biological effects of acridines, appeared to be due to a photodynamic action analogous to that of acriflavine. Sodium azide or anaerobiosis prevented the light-activated effects of 3-azido-10-methylacridinium chloride despite the fact that the initial chemical breakdown of the azido derivative induced by light was not affected. Cells suspended in D2O demonstrated an enhanced response to 3-azido-10-methylacridinium chloride with irradiation. These results indicate that singlet oxygen mediates the light-activated biological effects of both acriflavine and 3-azido-10-methylacridinium chloride.     

Synthesis of 3,6-bis[H-Tyr/H-Dmt-NH(CH2)m,n]-2(1H)pyrazinone derivatives: function of alkyl chain length on opioid activity

Dimeric opioid analogues linked to a pyrazinone platform, 3-[Tyr/Dmt-NH(CH2)m]-6-[Tyr/Dmt-NH(CH2)n]-2(1H)-pyrazinone (m, n=3 or 4), were synthesized. The Tyr-containing compound (m=4, n=3) exhibited mu-receptor affinity (K(i)mu; 7.58 nM) comparable to that of morphine, while the Dmt derivatives exhibited considerably higher affinity (K(i)mu; 0.021-0.051 nM) with corresponding agonism (IC50=1.79-4.93 nM). Interestingly one compound (m=4, n=3) revealed modest delta-opioid agonism; the converse analogue (m=3, n=4), however, was inactive in MVD assay.     

Evaluation of anti-inflammatory effect of synthetic 1,5-bis(4-acetoxy-3-methoxyphenyl)-1,4-pentadien-3-one,HB2

This work describes the synthesis and anti-inflammatory properties of a pentadienone derivative, HB2. The treatment with HB2 produced anti-oedematogenic, anti-inflammatory and antinociception without change locomotors performance. Finally, HB2 reduced the nitric oxide and prostaglandin E₂ production on RAW 264.7 cells stimulated with LPS without changing the cell viability. Taken together, our results show, for the first time, that HB2 can modulate the inflammatory response when administered to mice.     

Mixed-valency with cyanides as terminal ligands: Diruthenium(III,II) complexes with the 3,6-bis(2-pyridyl)-1,2,4,5-tetrazine bridge and variable co-ligands (CN vs. bpy or NH3)

New diruthenium complexes (PPN)₄[(NC)₄Ru(μ-bptz)Ru(CN)₄], (PPN)₄1, and [(bpy)₂Ru(μ-bptz)Ru(CN)₄], 2, (PPN⁺ = bis(triphenylphospine)iminium; bptz = 3,6-bis(2-pyridyl)-1,2,4,5-tetrazine; bpy = 2,2′-bipyridine), were synthesised and characterised by spectroscopic and electrochemical techniques. The comproportionation constant K_c = 10^7.0 of the mixed-valent species [(NC)₄Ru(μ-bptz)Ru(CN)₄]³⁻ as obtained by oxidation of 1⁴⁻ in CH₃CN is much lower than the K_c = 10^15.0 previously detected for [(H₃N)₄Ru(bptz)Ru(NH₃)₄]⁵⁺, reflecting the competition between CN⁻ and bptz for the π-electron density of the metals. Comparison with several other bptz-bridged diruthenium(II,III) complexes reveals an approximate correlation between K_c and the diminishing effective π acceptor capacity of the ancillary terminal ligands. In addition to the intense MLCT absorption at λ_max = 624 nm, the main IVCT (intervalence charge transfer) band of 1³⁻ was detected by spectroelectrochemistry at λ_max = 1695 nm (in CH₃CN; ε = 3200 M⁻¹ cm⁻¹). The experimental band width at half-height, Δν_1/2 = 2700 cm⁻¹, is slightly smaller than the theoretical value Δν_1/2 = 3660 cm⁻¹, calculated from the Hush approximation for Class II mixed-valent species. In agreement with comparatively moderate metal-metal coupling, the mixed-valent intermediate 1³⁻ was found to be EPR silent even at 4 K. The unsymmetrical mixed-valent complex [(bpy)₂Ru^II(μ-bptz)Ru^III(CN)₄]⁺, obtained in situ by bromine oxidation of 2 in CH₃CN/H₂O, displays a broad NIR absorption originating from an IVCT transition at λ_max = 1075 nm (ε ≈ 1000 M⁻¹ cm⁻¹, Δν_1/2 ≈ 4000 cm⁻¹). In addition, the lifetime of the excited-state of the mononuclear precursor complex [Ru(bptz)(CN)₄]²⁻ was measured in H₂O by laser flash photolysis; the obtained value of τ = 19.6 ns reveals that bptz induces a metal-to-ligand electronic delocalisation effect intermediate between that induced by bpy and bpz (bpz = 2,2′-bipyrazine) in analogous tetracyanoruthenium complexes.     

Synthesis, characterization and DNA binding studies of a zinc complex with 2,6-bis(benzimidazol-2-yl) pyridine

A zinc(II) complex having planar tridentate ligand, bzimpy, where bzimpy is 2,6-bis(benzimidazol-2-yl) pyridine was synthesized and characterized by UV, NMR, infrared spectroscopy and fluorescence spectra. The zinc complex acts as dibasic acids, in which N-H protons on benzimidazole moieties are responsible for a deprotonation site. Both the absorption spectra and reduction potentials are strongly dependent on the solution pH, which leads to the basis of a proton-induced molecular switch. The binding of this complex with calf thymus DNA has been investigated by absorption, luminescence titrations and viscosity measurements. The results suggest that the zinc(II) complex intercalates into DNA base pairs via the ligand bzimp.     

N-Aroyl-3,5-bis(benzylidene)-4-piperidones: a novel class of antimycobacterial agents

A number of 3,5-bis(benzylidene)-4-piperidones 1 and some N-4-(2-aminoethoxy)phenylcarbonyl analogs 3-6 display excellent in vitro antimycobacterial properties. In particular, 1c and 6d are potent antimycobacterials which are well tolerated in mice and are identified as important lead molecules. The nature of both the benzylidene aryl rings and the terminal basic groups which affect the antimycobacterial potencies and the absence of neurotoxic side effects were identified. Several representative compounds stimulated respiration in mitochondria isolated from rat liver and this effect was not caused by the swelling of these organelles. Various guidelines for the creation of further related novel antimycobacterial agents are provided.     

Crystal structures of heptakis(2,6-di-O-tert-butyldimethylsilyl)cyclomaltoheptaose, heptakis(2-O-methyl-3,6-di-O-tert-butyldimethylsilyl)cyclomaltoheptaose and heptakis(2-O-methyl)cyclomaltoheptaose

Crystal structures of heptakis(2,6-di-O-tert-butyldimethylsilyl)cyclomaltoheptaose, heptakis(2-O-methyl-3,6-di-O-tert-butyldimethylsilyl)cyclomaltohep taose and heptakis(2-O-methyl)cyclomaltoheptaose were determined from X-ray diffraction patterns obtained for single crystals of the title compounds grown from ethyl acetate and ethanol, respectively, as solvent. The crystal structures prove conclusively that quantitative migration of the tert-butyldimethylsilyl group from the 2-O- to the 3-O-position [D. Icheln, B. Gehrcke, Y. Piprek, P. Mischnick, W.A. Konig, M.A. Dessoy, A.F. Morel, Carbohydr. Res., 280 (1996) 237-250] was achieved during methylation of heptakis(2,6-di-O-tert-butyldimethylsilyl)cyclomaltoheptaose by iodomethane-sodium hydride.     

Free radicals in quinone containing antitumor agents. The nature of the diaziquone (3,6,-diaziridinyl-2,5-bis(carboethoxyamino)-1,4-benzoquinone) free radical

The enzymatically generated free radical of the antitumor agent diaziquone is analyzed with the help of two analogs where either the aziridine rings (RQ14) or the carboethoxyamino groups (RQ2) were substituted by chlorine atoms. The hyperfine couplings observed in the diaziquone free radical are due to the nitrogens in the aziridine group. Unresolved coupling and hindered rotation contribute to line broadening. We find that diaziquone free radicals are more stable than RQ14 but less stable than RQ2 free radicals. The reason for this is that the carboethoxyamino groups make the aromatic ring unstable, while the aziridines contribute to its stability. The free radical observed in diaziquone is in all probability that of the parent compound and not that of an intermediate metabolite.     

A bursal pentapeptide (BPP-I), a novel bursal-derived peptide, exhibits antiproliferation of tumor cell and immunomodulator activity

The bursa of Fabricius (BF) is the central humoral immune organ unique to birds. Here, we isolated a novel bursal pentapeptide I (BPP-I), LGPGP, from BF. BPP-I could play inhibition effect on MCF-7 but not on CEF or Vero cell proliferation in vitro, and enhance antitumor factor p53 protein expression. Also, BPP-I stimulated antibody production in a dose-dependent manner in hybridoma cell. Furthermore, BPP-I could induce various immune responses in mice immunization experiments, including increase antibody production and cytokines IL-4 and IFN-γ level, and induce T-cell immunophenotyping. These results suggest that BPP-I is a potential immunomodulator of antitumor and immunity. The study could provide some novel insights on the probable candidate reagent for the antitumor and immune improvement.     

3,5-bis(phenylmethylene)-1-(N-arylmaleamoyl)-4-piperidones: a novel group of cytotoxic agents

A series of novel 3,5-bis(phenylmethylene)-1-(N-arylmaleamoyl)-4-piperidones 3 have been synthesized which displayed potent cytotoxicity towards human Molt 4/C8 and CEM T-lymphocytes as well as murine P388 and L1210 leukemic cells. In contrast, the related N-arylmaleamic acids 4 possessed little or no cytotoxicity in these four screens. Molecular modeling revealed certain interplanar and bond angles and interatomic distances which were perceived to contribute to the observed bioactivity as well as providing suggestions for future structural modifications of the piperidones 3. Evaluation of representative compounds in series 3 and 4 on the activity of human N-myristoyltransferase revealed that, at the maximum concentration utilized, namely 250 microM, only weak inhibiting properties were displayed by some of the compounds in series 4. Various members of series 3 and 4 were well tolerated in mice.     

E,E,E-1-(4-Arylamino-4-oxo-2-butenoyl)-3,5-bis(arylidene)-4-piperidones: a topographical study of some novel potent cytotoxins

A series of E,E,E-3,5-bis(arylidene)-1-(4-arylamino-4-oxo-2-butenoyl)-4-piperidones 4 (phenylidene) and 5 (4-nitrophenylidene) were prepared in order to explore the structural features of the N-acyl group which affects the cytotoxic potency. Evaluation toward human Molt 4/C8 and CEM T-lymphocytes revealed that many of the IC(50) figures were submicromolar and lower than melphalan. Marked inhibitory potencies toward murine leukemia L1210 cells were also noted. When evaluated against a panel of human tumor cell lines, three representative compounds in series 4 displayed selective toxicity to leukemia and colon cancer cell lines and were significantly more potent than the reference drug melphalan. Molecular modeling of representative compounds in both series 4 and the analogs, in which the configuration of the olefinic double bond was changed from E to Z (series 3), revealed that the torsion angles of the arylidene aryl rings and locations of the terminal arylaminocarbonyl groups may have contributed to the greater cytotoxic properties displayed in 3. Compounds 4c (3,4-dichlorophenylamino), d (4-methylphenylamino) and 5c (3,4-dichlorophenylamino), d (4-methylphenylamino) inhibited the activity of human N-myristoyltransferase by approximately 50% at concentrations of 50-100 microM. The compounds in series 4 and 5 were well tolerated in a short-term toxicity study in mice.     

Induction of (2'-5')An-dependent endoribonuclease activity by interferon in cultured guinea pig macrophages

By a radiobinding assay, an affinity labeling technique, and a column procedure which quantitates mRNA intactness, evidence is presented on the induction of the (2'-5')An-dependent endoribonuclease by interferon in cultured guinea pig macrophages.     

Synthesis, characterization and antitumor activity of copper(II) complex with nicotinamido-4-bis(2-chloroethyl)aminobenzaldimine.

Nicotinamido-4-bis(2-chloroethyl)aminobenzaldimine (NBAB) was synthesized and characterized by elemental analysis, IR and 1H NMR spectra. The complex of Cu(NBAB)2(NO3)2 was prepared in ethanol and characterized by elemental analysis, conductivity, cyclic voltammetry, IR, UV-Vis, fluorescence, CD and EPR spectra. The characteristic data suggest that the complex has an elongated octahedral structure, and NBAB behaves as bidentate in the keto form. The antitumor activities of NBAB and the complex against L1210 murine leukemia and K562 were investigated with both the MTT method and the colony formation test. The results in vitro indicate that antitumor activities of NBAB are superior to 2,2'-chlorodiethylamine hydrochloride (nitrogen mustard) for L1210, and inferior to nitrogen mustard for K562, but the antitumor activities of the complex for both cell lines are superior to nitrogen mustard.     

Specificity protein 1 is a novel target of 2, 4-bis (p-hydroxyphenyl)-2-butenal for the suppression of human oral squamous cell carcinoma cell growth

Background

The Maillard reaction is a chemical reaction occurring between a reducing sugar and an amino acid, generally requiring thermal processing. Maillard reaction products (MRPs) have antioxidant, antimutagenic, and antibacterial effects though 2,4-bis (p-hydroxyphenyl)-2-butenal (HPB242), a fructose-tyrosine MRP, appears to inhibit proliferation of cancer cells, its mechanism of action has not been studied in detail. The purpose of this study was to investigate the anti-proliferative effects of 2,4-bis (p-hydroxyphenyl)-2-butenal (HPB242) on two oral squamous cell carcinoma (OSCC) cell lines, HN22 and HSC4, through regulation of specificity protein 1 (Sp1).

Results

HPB242 treatment dramatically reduced the cell growth rate and apoptotic cell morphologies. Sp1 was significantly inhibited by HPB242 in a dose-dependent manner. Furthermore, cell cycle regulating proteins and anti-apoptotic proteins, which are known as Sp1 target genes, were altered at the molecular levels. The key important regulators in the cell cycle such as p27 were increased, whereas cell proliferation- and survival-related proteins such as cyclin D1, myeloid leukemia sequence 1 (Mcl-1) and survivin were significantly decreased by HPB242 or suppressed Sp1 levels, however pro-apoptotic proteins caspase3 and PARP were cleaved in HN22 and HSC4.

Conclusions

HPB242 may be useful as a chemotherapeutic agent for OSCC for the purpose of treatment and prevention of oral cancer and for the improvement of clinical outcomes.